4.6. DCF Assay for Oxidative Stress Determination

To quantify intracellular ROS, a ROS assay kit (OxiSelectTM, Cell Biolabs, Inc., USA) was used. This assay is based on the cell permeable fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), which diffuses into cells and is deacetylated by intracellular esterases to the nonfluorescent 2′,7′-dichlorodihydrofluorescein (DCFH). In the presence of ROS, DCFH is rapidly oxidized to the highly fluorescent 2′,7′-dichlorodihydrofluorescein (DCF). The fluorescence intensity is proportional to the intracellular ROS levels.

According to the manufacturer’s protocol, cells were cultured at a density of 1 × 104 cells/well in a black 96-well plate and incubated overnight. Subsequently, media were removed, and cells were washed twice gently with DPBS (14190, GIBCO, Invitrogen, Carlsbad, CA, USA) and incubated with DCFH-DA/media solution for 30 min in the dark at 37 °C. Then, after removing the solution and washing the cells with DPBS, the DCFH-DA-loaded cells were exposed to TNFα (20 ng/mL) and 10 nm AgNPs (100 µg/mL) or 200 nm AgNPs (100 µg/mL) separately and together for 24 h. Parallel sets of wells containing DCFH-DA-loaded cells without any further exposure were used as a negative control. Another set of DCFH-DA-loaded cells were exposed to hydrogen peroxide (H₂O₂) and used as a positive control. The fluorescence of DCF was measured at regular intervals at an excitation/emission wavelength of 480 nm/530 nm using a fluorometric plate reader (Microplate Fluorometer, Twinkle LB 970, BERTHOLD TECHNOLOGIES, BadWildbad, Germany). The amounts of produced DCF were calculated based on a DCF standard curve.